Recombinant dimeric envelope vaccine against flaviviral infection

ABSTRACT

The present invention discloses and claims vaccines containing, as an active ingredient, a secreted recombinantly produced dimeric form of truncated flaviviral envelope protein. The vaccines are capable of eliciting the production of neutralizing antibodies against flaviviruses. The dimeric forms of truncated flaviviral envelope protein are formed 1) by directly linking two tandem copies of 80% E in a head to tail fashion via a flexible tether; 2) via the formation of a leucine zipper domain through the homodimeric association of two leucine zipper helices each fused to the carboxy terminus of an 80% E molecule; or 3) via the formation of a non-covalently associated four-helix bundle domain formed upon association of two helix-turn-helix moieties each attached to the carboxy terminus of an 80% E molecule. All products are expressed as a polyprotein including prM and the modified 80% E products are secreted from  Drosophila melanogaster  Schneider 2 cells using the human tissue plasminogen activator secretion signal sequence (tPA L ). Secreted products are generally more easily purified than those expressed intracellularly, facilitating vaccine production. One embodiment of the present invention is directed to a vaccine for protection of a subject against infection by dengue virus. The vaccine contains, as active ingredient, the dimeric form of truncated envelope protein of a dengue virus serotype. The dimeric truncated E is secreted as a recombinantly produced protein from eucaryotic cells. The vaccine may further contain portions of additional dengue virus serotype dimeric E proteins similarly produced. Another embodiment of the present invention is directed to methods to utilize the dimeric form of truncated dengue envelope protein for diagnosis of infection in individuals at risk for the disease. The diagnostic contains, as active ingredient, the dimeric form of truncated envelope protein of a dengue virus serotype. The dimeric truncated E is secreted as a recombinantly produced protein from eucaryotic cells. The diagnostic may further contain portions of additional dengue virus serotype dimeric E proteins similarly produced.

[0001] This is a continuation-in-part of application Ser. No.08/904,227, filed Jul. 31, 1997, which is incorporated herein in itsentirety.

TECHNICAL FIELD

[0002] This invention relates to protection against and diagnosis offlaviviral infection. More specifically, the invention concernsrecombinantly produced dimers of truncated flaviviral envelope proteinsecreted as mature proteins from eucaryotic cells and which induce hightiter virus neutralizing antibodies believed to be important inprotection against flaviviral infection and which are useful indiagnosis of infection by the virus.

BACKGROUND ART

[0003] The four serotypes of dengue virus (DEN-1, DEN-2, DEN-3, andDEN-4) belong to the family Flaviviridae which also includes theJapanese encephalitis virus (JE), Tick-borne encephalitis virus (TBE),West Nile virus (WN), and the family prototype, Yellow fever virus (YF).Flaviviruses are small, enveloped viruses containing a single,positive-strand, genomic RNA. The envelope of flaviviruses is derivedfrom the host cell membrane and is decorated with virally-encodedtransmembrane proteins membrane (M) and envelope (E). While mature Eprotein and the precursor to M, prM, are glycosylated, the much smallermature M protein is not. The E glycoprotein, which is the largest viralstructural protein, contains functional domains responsible for cellsurface attachment and intraendosomal fusion activities. It is also amajor target of the host immune system, inducing virus neutralizingantibodies, protective immunity, as well as antibodies which inhibithemagglutination.

[0004] Dengue viruses are transmitted to man by mosquitoes of the genusAedes, primarily A. aegypti and A. albopictus. The viruses cause anillness manifested by high fever, headache, aching muscles and joints,and rash. Some cases, typically in children, result in a more severeforms of infection, dengue hemorrhagic fever and dengue shock syndrome(DHF/DSS), marked by severe hemorrhage, vascular permeability, or both,leading to shock. Without diagnosis and prompt medical intervention, thesudden onset and rapid progression of DHF/DSS can be fatal.

[0005] Flaviviruses are the most significant group ofarthropod-transmitted viruses in terms of global morbidity and mortalitywith an estimated one hundred million cases of dengue fever occurringannually (Halstead, 1988). With the global increase in population andurbanization especially throughout the tropics, and the lack ofsustained mosquito control measures, the mosquito vectors of flavivirushave distributed throughout the tropics, subtropics, and some temperateareas, bringing the risk of flaviviral infection to over half theworld's population. Modem jet travel and human emigration havefacilitated global distribution of dengue serotypes, such that nowmultiple serotypes of dengue are endemic in many regions. Accompanyingthis in the last 15 years has been an increase in the frequency ofdengue epidemics and the incidence of DHF/DSS. For example, in SoutheastAsia, DHF/DSS is a leading cause of hospitalization and death amongchildren (Hayes and Gubler, 1992).

[0006] The flaviviral genome is a single strand, positive-sense RNAmolecule, approximately 10,500 nucleotides in length containing short 5′and 3′ untranslated regions, a single long open reading frame, a 5′ cap,and a nonpolyadenylated 3′ terminus. The complete nucleotide sequence ofnumerous flaviviral genomes, including all four DEN serotypes and YFvirus have been reported (Fu et al., 1992; Deubel et al., 1986; Hahn etal., 1988; Osatomi et al., 1990; Zhao et al., 1986; Mackow et al., 1987;Rice et al., 1985). The ten gene products encoded by the single openreading frame are translated as a polyprotein organized in the order,capsid (C), premembrane/membrane (prM/M), envelope (E), nonstructuralprotein (NS) 1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5 (Chambers, et al.1990). Processing of the encoded polyprotein is initiatedcotranslationally, and full maturation requires both host andvirally-encoded proteases. The sites of proteolytic cleavage in the YFvirus have been determined by comparing the nucleotide sequence and theamino terminal sequences of the viral proteins. Subsequent to initialprocessing of the polyprotein, prM is converted to M during viralrelease (Wengler, G. et al. J Virol (1989) 63:2521-2526) and anchored Cis processed during virus maturation (Nowak et al. Virology (1987)156:127-137).

[0007] While all dengue viruses are antigenically related, antigenicdistinctions exist which define the four dengue virus serotypes.Infection of an individual with one serotype does not apparently providelong-term immunity against the other serotypes. In fact, secondaryinfections with heterologous serotypes are becoming increasinglyprevalent as multiple serotypes co-circulate in a geographic area. Ingeneral, primary infections elicit mostly IgM antibodies directedagainst type-specific determinants. On the other hand, secondaryinfection by a heterologous serotype is characterized by IgG antibodiesthat are flavivirus cross-reactive. Dengue virus vaccine development iscomplicated by the observation that immunity acquired by infection withone serotype may in fact enhance pathogenicity by dengue virus of othertypes. Halstead (1982) demonstrated that anti-dengue antibodies canaugment virus infectivity in vitro, and proposes that serotypecross-reactive, non-neutralizing antibodies to E enhance infection invivo, resulting in DHF/DSS (Halstead, 1981). This viewpoint is nothowever, universally accepted (Rosen, 1989). For example, Kurane et al.(1991) proposed that dengue serotype-cross-reactive CD4⁺ CD8⁻ cytotoxicT cells (CTLs) specific for NS3 may contribute to the pathogenesis ofDHF/DSS by producing IFN-γ and by lysing dengue virus-infectedmonocytes. Recent evidence demonstrating that CTLs specific for E arenot serotype-cross-reactive may suggest that use of E subunit vaccineswould not induce the potentially harmful cross-reactive CTL response(Livingston et al., 1994). Regardless of the mechanism for enhancedpathogenicity of a secondary, heterologous dengue viral infection,strategies employing a tetravalent vaccine should avoid suchcomplications. Helpful reviews of the nature of the flaviviral diseases,the history of attempts to develop suitable vaccines, and structuralfeatures of flaviviruses in general as well as the molecular structuralfeatures of the envelope protein of flaviviruses are available (Halstead1988; Brandt 1990; Chambers et al., 1990; Mandl et al., 1989; Henchaland Putnak, 1990; Putnak 1994; Rey et al., 1995).

[0008] Although many approaches to dengue virus vaccines have beenpursued, there is no acceptable vaccine currently available. Untilrecently, the low titer of dengue virus grown in culture has made akilled vaccine impractical, and candidate live-attenuated dengue virusvaccine strains tested to date have proven unsatisfactory (see, e.g.,Eckels et al., 1984; Bancroft et al., 1984; McKee et al., 1987),although live attenuated candidate vaccine strains continue to bedeveloped and tested (Hoke et al., 1990; Bhamarapravati et al., 1987).The construction of several full-length infectious flavivirus clones(Rice et al., 1989; Lai et al., 1991; Sumiyoshi et al., 1992) hasfacilitated studies aimed at identifying the determinants of virulencein flaviviruses (Bray and Lai, 1991; Chen et al., 1995; Kawano et al.,1993). However, these studies are in preliminary stages and littleinformation on virulence has been obtained. A similar approach tovaccine development in the poliovirus system, while extremelyinformative, has taken years.

[0009] In the absence of effective live attenuated or killed flavivirusvaccines, a significant effort has been invested in the development ofrecombinant, flaviviral subunit or viral-vectored vaccines. Many of thevaccine efforts which use a recombinant DNA approach have focused on theE glycoprotein. This glycoprotein is a logical choice for a subunitvaccine as it is exposed on the surface of the virus and is believed tobe responsible for eliciting protective immunity as monoclonalantibodies directed against purified flaviviral E proteins areneutralizing in vitro and some have been shown to confer passiveprotection in vivo (Henchal et al., 1985; Heinz et al., 1983; Mathews etal., 1984; Hawkes et al., 1988; Kimuro-Kuroda and Yasui, 1988).

[0010] Although the primary amino acid sequence of flaviviral Eglycoproteins are variable (45-80% identity), all have twelve conservedcysteine residues, forming six disulfide bridges, and nearlysuperimposable hydrophilicity profiles suggesting that they probablyhave similar secondary and tertiary structures. Recently, the structureof a soluble fragment of the Tick-borne encephalitis (TBE) virusenvelope glycoprotein was solved at 2 Å resolution (Rey et al., 1995).This analysis demonstrated that the envelope glycoprotein in its nativeform is a homodimer which presumably extends parallel to the virionsurface. This dimer is formed by an anti-parallel association of the twoenvelope glycoproteins stabilized by polar interactions along thecentral region of the dimer, and by non-polar interactions at either end(FIG. 1). The dimer is slightly curved relative to the virion surface,perhaps conforming to the shape of the lipid envelope. The convex,external face contains the major immunogenic sites and the carbohydrateside chains. The carboxy terminus extends from the concave internal facedown toward the membrane. Based upon sequence alignments andconservation of cysteine residues involved in disulfide bridges, theauthors suggest that the TBE structure serves as a good model for allflavivirus envelopes. Therefore, recombinant soluble dengue E expressedas a dimer might induce a more potent antiviral response than monomericE because it more closely resembles the natural envelope glycoprotein.

[0011] Bioenvelope glycoproteins vary widely in primary, secondary,tertiary, and quaternary structure. Functional similarity does notnecessarily imply structural similarity. To demonstrate the type ofvariation seen in viral envelope glycoproteins one need look no furtherthan the structures of HIV envelope, Tick Borne Encephalitis (TBE) virusenvelope (a flavivirus very similar to dengue), influenza virushemagglutinin glycoprotein, and Semliki Forest Virus envelope (SFV; analpha virus). In terms of primary structure, the envelope glycoproteinstend to be the most highly divergent of any viral gene and thus minimalsequence similarity exists even within groups of closely relatedviruses. As one looks at highly divergent viruses (e.g. HIV and TBE ordengue) the sequence similarity is almost non-existent. In addition,they vary significantly in terms of secondary, tertiary, and quaternarystructure as well. As illustrated in Kwong, P. D. et al. Nature (1998)393:648-659, the structure of the HIV gp120 envelope glycoprotein isquite globular in nature and in fact does not include a transmembranedomain. The membrane anchor function of the HIV envelope glycoprotein isprovided by another protein, gp41 which associates non-covalently as aheterodimer with the gp120 protein maintaining its association with themembrane. In contrast, the structure of the flavivirus TBE envelopeglycoprotein (Rey, F. A. et al. Nature (1995) 375:291-298) demonstratedthat it exhibits an elongated structure. However, in contrast to otherviral envelope glycoproteins which also have an elongated structure(e.g. influenza virus hemagglutinin discussed below) the elongatedstructure lies parallel to the membrane in a rather flat presentation.In fact, the flavivirus envelope exists on the surface of the membraneas a homodimer with head to tail orientation of the two monomers and isanchored in the membrane by its own transmembrane domain. The structureof the envelope glycoproteins of influenza virus (hemagglutinin andneuraminidase), while also elongated in form, exist as spikes protrudingfrom the membrane and include unique structural features such as a hingeregion (Reviewed in Fields, B. N. and D. M. Knipe (eds.) Virology,2^(ed)., Raven Press, NY, 1990). The hemagglutinin spikes are formed bythe association of three monomers in a triple-stranded coiled-coilstructure markedly different from the head to tail dimer form of the TBEenvelope. Finally, although the alphaviruses are relatively closelyrelated to the flaviviruses, the structure of an alphavirus envelopeglycoprotein also varies significantly from the structure described forflaviviruses (Helenius, A. Cell (1995) 81:651-653). The SFV envelopeglycoproteins have been shown to form spikes which project 80 nm fromthe membrane surface and consist of three E1-E2 pairs. Thus, even forrelatively closely related viruses, the envelope glycoproteins, whileserving the same function, have markedly different structuralproperties.

[0012] These markedly different primary, secondary, tertiary, andquaternary structures affect heterologous expression characteristics. Infact, in contrast to HIV envelope glycoprotein which is expressed atreasonable efficiency in both the Chinese Hamster Ovary (CHO) cellexpression system (Berman et al. J Virol (1989) 63:3489-98) and theDrosophila cell expression system (Culp et al.), the dengue virusenvelope glycoprotein is not efficiently expressed in CHO but isefficiently expressed in the Drosophila system. Expression levels ofdengue envelope in CHO being less than 0.1 mg/L.

[0013] Recombinant flavivirus E glycoprotein has been expressed inseveral systems to date (See Putnak, 1994 for recent review). In generalthe systems have proven unsatisfactory for production of acost-effective flavivirus vaccine due to limitations in antigen quality,quantity, or both. The following paragraphs highlight the majorflavivirus vaccine efforts and summarize the results obtained to date.

[0014] Most efforts using Escherichia coli have yielded poor immunogenincapable of eliciting neutralizing antibodies in mice. This may reflectnon-native conformation of flavivirus proteins expressed by bacteria andthe necessity to process the viral proteins through the secretionpathway in order to achieve proper disulfide bond formation andglycosylation. Expression of dengue proteins using the eucaryotic yeastsSaccharomyces cerevisiae and Pichia pastoris results in less thandesirable quantities of immunogenic recombinant product obtained. Theexpression levels of dengue E achieved in these systems are well belowthat which would be required to produce a cost-effective flavivirusvaccine. (John Ivy et al., unpublished data. Expression of 80% E in theabove-mentioned yeast systems and fungal systems (Neurospora crassa)gave products that were highly glycosylated (contain extensive highmannose chains) which interferes with immunogenicity. Also, the yieldswere quite low (ranging from about 10-100 ng/ml (despite the ability ofthese systems to produce high yields generally).)

[0015] Attempts to express 80% E in the Chinese Hamster Ovary (CHO)cells expression system were particularly disappointing. Predictionsthat this mammalian expression system would be ideal for a Flavivirusenvelope expression (since this is a virus which normally infectsmammals and the system supports all the necessary post-translationalmodifications required to get native confirmation, were wrong. In factthe yields were poorest of any system (less than 0.1 μg/ml) and theDengue envelope gene was completely unstable in this expression system.

[0016] Use of the baculovirus expression system for flavivirus subunitvaccine production has met with limited success (Reviewed in Putnak,Modern Vaccinology, 1994). In contrast to the high expression levelsreported for various heterologous proteins in the baculovirus system,the levels of expression of flavivirus structural proteins were quitelow (e.g. 5-10 μg DEN-2 E/10⁶ cells; Deubel et al., 1991), andreactivity against a panel of anti-flaviviral monoclonal antibodies(MAbs) indicated that many conformationally sensitive epitopes were notpresent (Deubel et al., 1991). This suggests that folding of recombinantE produced in the baculovirus system may differ from the natural viral Eprotein. Furthermore, immunization with baculovirus-expressedrecombinant envelope protein from DEN-1 (Putnak et al., 1991), JapaneseEncephalitis virus (McCown et al., 1990), or Yellow Fever virus (Despreset al., 1991) failed to elicit substantial titers of virus neutralizingantibodies or protection against viral challenge in mice.

[0017] Several reports have described Vaccinia flavivirus recombinantsexpressing envelope proteins as part of a polyprotein. The mostconsistently successful results in vaccinia expression of flaviviralproteins have been obtained co-expressing prM and E. Mice immunized withrecombinant vaccinia expressing Japanese Encephalitis (JE) virus prM andE developed higher neutralizing antibody titers and survived higherchallenge doses of virus (>10,000 LD₅₀; Konishi et al., 1992) than miceimmunized with recombinant vaccinia virus expressing E alone (>10 LD₅₀;Mason et al., 1991). Similarly, mice immunized with a vaccinia-YellowFever (YF) virus recombinant expressing prM-E were protected from viruschallenge at levels equivalent to that of the attenuated YFV-17Dvaccine, while vaccinia-YF virus recombinants expressing E-NS 1,C-prM-E-NS 1, or NS1 failed to protect mice (Pincus et al., 1992).Vaccinia-DEN-1 recombinants expressing prM-E elicited neutralizing andhemagglutination inhibiting antibodies in mice, while recombinantsexpressing DEN-1 C-prM-E-NS1-NS2a-NS2b elicited no E-specific immuneresponse (Fonseca et al., 1994).

[0018] Coordinate synthesis of prM and E appears to be important toobtain the native conformation of E. Expression of E in the absence ofprM may result in a recombinant product that presents a different set ofepitopes than those of the native virion (Konishi and Mason 1993; Heinzet al., 1994; Matsuura et al., 1989). Epitope mapping of the E expressedwith prM suggests that the co-expressed protein more closely resemblesthe native virus. As prM and E appear to form heterodimers during viralmaturation and E undergoes an acid pH-induced conformational change,Heinz et al. (1994) has suggested the association of prM and E isrequired to prevent irreversible pH-induced conformational changesduring transit through the secretory pathway. However, it has been shownthat carboxy-truncated forms of flavivirus E expressed in the absence ofprM elicit protection from challenge (Men et al., 1991; Jan et al.,1993; Coller et al., in preparation), suggesting expression of E in theabsence of prM can result in the display of protective epitopes.

[0019] Within the last ten years an alternative eucaryotic expressionsystem which uses the Drosophila melanogaster Schneider 2 (S2) cell linehas been developed and used to efficiently express the envelopeglycoprotein of Human Immunodeficiency Virus (Ivey-Hoyle et al., 1991;Culp et al., 1991; van der Straten et al., 1989). We have applied thissystem to production of recombinant flavivirus subunit polypeptides andhave found the system can easily produce 20-30 mg of recombinant proteinper liter of medium (unpublished). The recombinant product we havefocused most of our efforts on is a soluble form of flaviviral E, whichis truncated at the carboxy-terminal end resulting in a polypeptidewhich represents approximately 80% of the full-length E molecule (aminoacids 1-395; 80% E). We have expressed 80% E as a single open-readingframe with prM to enhance proper folding and secretion as describedabove. The expression levels achieved using this combination ofexpression system and recombinant DNA construct far exceed thoseachieved in other systems and does provide a cost-effective source offlaviviral antigen for vaccine production. In addition, we havedemonstrated that the recombinant 80% E product secreted by these cellsis capable of inducing neutralizing antibodies and protection in mice(Coller et al., in preparation.) In two instances, however, applicantsfailed in their attempt to produce envelope glycoproteins in theDrosophila expression system. First, a 100-amino acid polypeptide whichis a unique domain (Domain B; amino acids 296-395 of DEN-2E) within the80% E molecule was expressed poorly in the Drosophila expression system.The expression levels for Domain B were significantly lower (less than 1mg/l) than those achieved with 80% E (approximately 15 mg/l). Domain Bwas the most highly expressed polypeptide in S. cereviseae and P.pastoris which we evaluated with expression levels up to 575 mg/l forDomain B expressed in P. pastoris (compared to expression levels ofapproximately 1 mg/l for 80% E). Second, a truncated version of themeasles hemagglutinin protein (90% HA) was expressed and secreted atvery low levels in the Drosophila expression system (about 0.5 mg/l).Like dengue, measles has been refractory to stable expression in manysystems (Hirano, A. et al. “Generation of mammalian cells expressingstably measles virus proteins via bicistronic RNA,” Journal ofVirological Methods (1991) 33:135-147).

[0020] The two examples above show that protein expression is highlyunpredictable (Goeddel, D. V. “Systems for Heterologous GeneExpression,” in Methods in Enzymology, Vol. 185, pp. 3—Academic Press,Inc., 1990). In this case, protein expression is further complicated bythe complexity of expressing bioenvelope glycoproteins (Mustilli, A. C.et al. “Comparison of secretion of a hepatitis C virus glycoprotein inSaccharomyces cerevisiae and Kluyveromyces lactis,” Res Microbiol (1999)150:179-187).

[0021] Over the past approximately eight years of research relating todengue 80% E, the assignee of the present application has spent over$6.5 million to arrive at the invention.

[0022] While the use of the combination of Drosophila S2 cells andprM80% E has allowed significant progress towards the production of aneffective flavivirus vaccine, the ability of a small polypeptide, withlimited antigenic complexity, to induce long term, protective immunityin a large, outbred population may be limited. Numerous studies havedemonstrated that immunogenicity is directly related both to the size ofthe immunogen and to the antigenic complexity of the immunogen. Thus, ingeneral, larger antigens make better immunogens. In addition, thestructure of TBE envelope protein was recently solved (Rey et al., 1995)and this analysis revealed that the native form of E protein found onthe surface of the virion is a homodimer (FIG. 1). Our recombinantflaviviral E protein discussed above is monomeric and therefore is notidentical to the natural viral E protein. Thus, in an attempt to producea recombinant flavivirus vaccine with enhanced immunogenicity weengineered several constructs designed to promote dimerization of thesoluble 80% E which is so efficiently produced in the Drosophila cells.By enhancing dimerization we increase the potency of the vaccine byincreasing the structural similarity to native, virally expressed E, aswell as by increasing the size and antigenic complexity of theimmunogen.

[0023] Several of the approaches we have adopted to enhance dimerizationof soluble 80% E were originally developed for antibody engineering.Flexible peptide linkers have been used to link the variable heavy andvariable light chain polypeptides in the engineering of single chainFv's (scFv; Huston et al., 1988; Bird et al., 1988). These linkers,which are often repeated GlyGlyGlyGlySer (Gly4Ser) units, exhibitlimited torsional constraints on the linked polypeptides, and thereforeoffer a reasonable option for covalently connecting the carboxy end ofone 80% E moiety to the amino terminus of the second 80% E moiety. Basedon the distance from the carboxy terminus of one subunit and the aminoterminus of the other in the crystal structure of TBE 80% E dimers (F.Heinz, personal communication), we designed a peptide linker, made uppredominantly of Gly4Ser repeats, to link the two 80% E molecules. Thelinker was designed to be slightly longer than the distance in thenative molecule, in order to avoid torsional constraint on theassociation of the two 80% E moieties.

[0024] The second and third approaches to engineer 80% E dimers usedstrategies developed to engineer self-associating scFv miniantibodies.For homodimer miniantibody expression, Pack et al. (1992; 1993)expressed the scFv as a fusion with a flexible linker hinge and one oftwo dimerization domains (FIG. 2). One dimerization domain was aparallel coiled-coil helix of a leucine zipper from the yeast GCN4 geneproduct (Landschulz et al., 1988; O'Shea et al, 1989). The other domainwas two alpha helices spaced by a sharp turn that associate to form ahomodimeric four-helix bundle (Ho and DeGrado, 1987). The hinge regionused to link the dimerization domains to the scFv was taken from anantibody hinge region to achieve maximum rotational flexibility. Whenthese antibody-hinge-helix constructs were expressed in E. coli,homodimer miniantibodies spontaneously formed and could be extractedfrom the soluble protein fraction of cell lysates. These antibodies wereindistinguishable from whole antibodies in functional affinity. Toexpress secreted 80% E that can spontaneously dimerize, we have usedthese dimerization domains connected to the 80% E domains by a flexibleGly4Ser tether.

DISCLOSURE OF THE INVENTION

[0025] The present invention discloses and claims vaccines containing,as an active ingredient, a secreted recombinantly produced dimeric formof truncated flaviviral envelope protein. The vaccines are capable ofeliciting the production of neutralizing antibodies against flavivirus.In the illustrations below, the dimeric forms of truncated flaviviralenvelope protein are formed 1) by directly linking two tandem copies of80% E in a head to tail fashion via a flexible tether; 2) via theformation of a leucine zipper domain through the homodimeric associationof two leucine zipper helices each fused to the carboxy terminus of an80% E molecule; or 3) via the formation of a non-covalently associatedfour-helix bundle domain formed upon association of two helix-turn-helixmoieties each attached to the carboxy terminus of an 80% E molecule. Allproducts are expressed as a polyprotein including prM and the modified80% E products are secreted from Drosophila melanogaster Schneider 2cells using the human tissue plasminogen activator secretion signalsequence (tPA₁). Secreted products are generally more easily purifiedthan those expressed intracellularly, facilitating vaccine production.

[0026] One embodiment of the present invention is directed to a vaccinefor protection of a subject against infection by a Flavivirus. Thevaccine contains, as active ingredient, the dimeric form of truncatedenvelope (E) protein of a flaviviral serotype, for example a denguevirus serotype. The dimeric truncated E is secreted as a recombinantlyproduced protein from eucaryotic cells. The vaccine may further containportions of additional flaviviral serotype dimeric E proteins similarlyproduced. A preferred embodiment of the present invention relates to avaccine for the protection of a subject against infection by a denguevirus. The vaccine contains a therapeutically effective amount of adimeric 80% E, where, the 80% E has been secreted as a recombinantlyproduced protein from eucaryotic cells, such as Drosophila cells.Further, the “80% E” refers in one instance to a polypeptide which spansfrom Met 1 to Gly 395 of the DEN-2 envelope protein. The sequencesdescribed in the present application represent the envelope protein fromdengue type 2 virus; three additional distinct dengue serotypes havebeen recognized. Therefore, “80% E” also refers to the correspondingpeptide region of the envelope protein of these serotypes, and to anynaturally occurring variants, as well as corresponding peptide regionsof the envelope (E) protein of other flaviviruses. For example,serotypes of dengue virus such as: DEN-1; DEN-2; DEN-3; and DEN-4, aswell as serotypes of: Japanese encephalitis virus (JE), Tick-borneencephalitis virus (TBE), West Nile virus (WN), and the familyprototype, Yellow fever virus (YF).

[0027] Other embodiments of the present invention are directed to threebasic approaches for the construction of dimeric 80% E molecules. (Seeinfra.) These include: linked 80% E dimer; 80% E ZipperI; 80% EZipperII; and 80% E Bundle.

[0028] Still other embodiments of the present invention are directed tovaccines containing truncated envelope protein of dimeric 80% E of morethan one serotype to form multivalent vaccines, (i.e., divalent,trivalent, tetravalent, etc.). For example, such embodiments of thepresent invention include: a vaccine containing a first dimeric 80% Eproduct of one flaviviral serotype and a second dimeric 80% E product ofa second flaviviral serotype, and a third dimeric 80% E product of athird flaviviral serotype and a fourth dimeric 80% E product of a fourthflaviviral serotype, as well as in combination with other dimeric 80% E,each of a separate serotype one from another, where all dimeric 80% Eshave been secreted as recombinantly produced protein from eucaryoticcells, such as Drosophila cells. It is considered that the presentinvention clearly includes vaccines that are comprised of multivalenttruncated envelope protein of dimeric 80% E, which embrace two, three,four or more serotypes. For example, these serotypes may include thefollowing dengue virus serotypes: DEN-1; DEN-2; DEN-3; and DEN-4, aswell as other flavivirus scrotypes of: Japanese encephalitis virus (JE),Tick-borne encephalitis virus (TBE), West Nile virus (WN), and thefamily prototype, Yellow fever virus (YF).

[0029] Additional embodiments of the present invention contemplatecompositions of antibodies consisting essentially of antibodiesgenerated in a mammalian subject administered an immunogenic amount of avaccine containing dimeric 80% E as well as containing a first dimeric80% E and a second dimeric 80% E, where both first and second dimeric80% E have been secreted as recombinantly produced protein fromeucaryotic cells, such as Drosophila cells. These vaccines could includemultivalent truncated envelope protein of dimeric 80% E, which embracetwo, three, four or more serotypes. These serotypes may include denguevirus serotypes: DEN-1; DEN-2; DEN-3; and DEN-4, as well as serotypesof: Japanese encephalitis virus (JE), Tick-borne encephalitis virus(TBE), West Nile virus (WN), and the family prototype, Yellow fevervirus (YF).

[0030] Still other embodiments of the present invention are drawn toimmortalized B cell lines, where the B cells have been generated inresponse to the administration to a mammalian subject of an immunogenicamount of a vaccine containing truncated envelope protein of dimeric 80%E of more than one serotype to form multivalent vaccines, (i.e.,divalent, trivalent, tetravalent, etc.). For example, such embodimentsof the present invention include: a vaccine containing a first dimeric80% E product of one flaviviral serotype and a second dimeric 80% Eproduct of a second flaviviral serotype, and a third dimeric 80% Eproduct of a third flaviviral serotype and a fourth dimeric 80% Eproduct of a fourth flaviviral serotype, as well as in combination withother dimeric 80% E, each of a separate serotype one from another, whereall dimeric 80% Es have been secreted as recombinantly produced proteinfrom eucaryotic cells, such as Drosophila cells. These vaccines couldinclude multivalent truncated envelope protein of dimeric 80% E, whichembrace two, three, four or more serotypes. These serotypes may includedengue virus serotypes: DEN-1; DEN-2; DEN-3; and DEN-4, as well asserotypes of: Japanese encephalitis virus (JE), Tick-borne encephalitisvirus (TBE), West Nile virus (WN), and the family prototype, Yellowfever virus (YF).

[0031] Further embodiments of the present invention are drawn tomonoclonal antibodies secreted by these immortalized B cell lines.

[0032] Still further embodiments of the present invention are drawn tomethods to protect a subject against a Flavivirus. These methods includethe step of administering in a suitable manner to a subject in need ofsuch protection an effective amount of a vaccine containing dimeric 80%E on a schedule optimum for eliciting such a protective immunoreactiveresponse.

[0033] Another embodiment of the present invention is directed tomethods to utilize the dimeric form of truncated flavivirus envelopeprotein for diagnosis of infection in individuals at risk for thedisease. The diagnostic contains, as active ingredient, the dimeric formof truncated envelope protein of a flavivirus serotype. The dimerictruncated E is secreted as a recombinantly produced protein fromeucaryotic cells. The diagnostic may further contain portions ofadditional flavivirus serotype dimeric E proteins similarly produced.

[0034] A preferred embodiment of the present invention relates to animmunodiagnostic for the detection of a Flavivirus, where theimmunodiagnostic contains, a dimeric 80% E that has been secreted as arecombinantly produced protein from eucaryotic cells, such as Drosophilacells. Specifically, a preferred embodiment of the present inventionrelates to an immunodiagnostic for the detection of a flavivirus.Embodiments of the present invention include immunodiagnostics for thedetection of a Flavivirus, where the immunodiagnostic contains, dimeric80% E of more than one serotype to form multivalent immunodiagnostics,(i.e., divalent, trivalent, tetravalent, etc.). For example, suchembodiments of the present invention include: an immunodiagnosticscontaining a first dimeric 80% E product of one flaviviral serotype anda second dimeric 80% E product of a second flaviviral serotype, and athird dimeric 80% E product of a third flaviviral serotype and a fourthdimeric 80% E product of a fourth flaviviral serotype, as well as incombination with other dimeric 80% E, each of a separate serotype onefrom another, where all of the dimeric 80% Es have been secreted asrecombinantly produced protein from eucaryotic cells, such as Drosophilacells.

[0035] The present invention includes the embodiments ofimmunodiagnostic kits for the detection of a Flavivirus, in a testsubject. These immunodiagnostic kits contain (a) dimeric 80% E, wherethe dimeric 80% E has been secreted as recombinantly produced proteinfrom eucaryotic cells, such as Drosophila cells; (b) a suitable solidsupport phase coated with dimeric 80% E; and (c) labeled antibodiesimmunoreactive to antibodies from the test subject.

[0036] Other embodiments of the immunodiagnostic kits of the presentinvention include multivalent dimeric 80% E of more than one serotype toform multivalent immunodiagnostics, (i.e., divalent, trivalent,tetravalent, etc.). For example, such embodiments of the presentinvention include: an immunodiagnostics containing a first dimeric 80% Eproduct of one flaviviral serotype and a second dimeric 80% E product ofa second flaviviral serotype, and a third dimeric 80% E product of athird flaviviral serotype and a fourth dimeric 80% E product of a fourthflaviviral serotype, as well as in combination with other dimeric 80% Eproducts, each of a separate serotype one from another, where all of thedimeric 80% E products have been secreted as recombinantly producedprotein from eucaryotic cells, such as Drosophila cells.

[0037] Further embodiments of the present invention relate tocompositions of matter, that include a vector host recombinant DNAexpression system, containing: (a) a suitable eucaryotic host cell; (b)a suitable recombinant DNA expression vector; (c) DNA encoding dimeric80% E, operably linked and under the control of a suitable promoter; and(d) where the DNA encoding dimeric 80% E is also operably linked to asecretory signal leader sequence. The present invention further includesembodiments of a vector host recombinant DNA system where the dimeric80% E is selected from the group consisting of: linked 80% E dimer; 80%E ZipperI; 80% E ZipperII; and 80% E Bundle. A preferred embodiment ofthe present invention relates to a vector host recombinant DNA systemwhere the eucaryotic host cell is a Drosophila cell.

[0038] Other compositions of matter embodied in the present inventioninclude DNA sequences encoding dimeric 80% E, specifically including DNAsequences encoding: linked 80% E dimer; 80% E ZipperI; 80% E ZipperII;and 80% E Bundle.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039]FIG. 1 is a drawing reproduced from Rey et al., showing thecrystal structure of the envelope protein of Tick Borne Encephalitisvirus.

[0040]FIG. 2 is a drawing reproduced from Pack et al., which shows twothe approaches used for miniantibody engineering applied to 80% E dimerformation.

[0041]FIG. 3 shows the partial nucleotide sequence and deduced aminoacid sequence of the genome of DEN-2 PR159/S1 strain.

[0042]FIG. 4 is a drawing illustrating the strategy used to generatecDNA encoding tandem copies of 80% E linked by a flexible tether.

[0043]FIG. 5 is a drawing illustrating the cloning strategy used tointroduce the carboxy-terminal portion of the first 80% E—linker—andamino terminal portion of the second 80% E molecule into a prM80% E cDNAclone.

[0044]FIG. 6 is a drawing illustrating the cloning strategy used tointroduce the linked tandem copies of 80% E into a Drosophila expressionvector.

[0045]FIG. 7 illustrates the cloning strategy used to introduceoligonucleotides encoding the leucine zipper and four-helix bundledimerization domains into the linked 80% E dimer cDNA clone.

[0046]FIG. 8 is a drawing illustrating the cloning strategy used tointroduce the cDNA fragments encoding Linked 80% E Dimer, 80% E ZipperI,80% E ZipperII, and 80% E Bundle into a Drosophila expression vector.

[0047]FIG. 9 shows the SDS-PAGE analysis of the expressed dimeric 80% Eproducts secreted from transfected S2 cells.

[0048]FIG. 10 demonstrates the glycosylation of the secreted dimeric 80%E products by SDS-PAGE analysis of endoglycosidase-digested 80% Edimers.

[0049]FIG. 11 demonstrates the application of immunoaffinity techniquesto purification of the secreted dimeric 80% E products.

MODES OF CARRYING OUT THE INVENTION

[0050] The invention provides, for the first time, a subunit vaccinewith increased immunogenicity that can be efficiently produced andsecreted using a recombinant expression system and that is effective ininducing a strong virus neutralizing response to flaviviruses. Althoughmany attempts have been made to obtain such a subunit vaccine, previousstudies were plagued with either low expression levels of an effectiveimmunogen or efficient production of an ineffective vaccine candidate.The present applicants have found that recombinantly-engineered, dimericforms of a carboxy-terminally truncated flaviviral envelope protein,corresponding to amino acids 1-395, are efficiently secreted by certainconvenient eucaryotic recombinant hosts, in a form that permitsprocessing to mimic the native conformation of the protein. Theefficient secretion of the proteins into the culture medium facilitatespurification. Furthermore, the secreted forms are able, especially whenadministered in the presence of adjuvant, to raise high titer virusneutralizing antibodies in animals. Thus, these proteins represents auseful component of a vaccine for protecting subjects against flaviviralinfection.

[0051] As used herein, “80% E” refers in one instance to a polypeptidewhich spans from Met 1 to Gly 395 of the DEN-2 envelope protein. Thesequences described in the present application represent the envelopeprotein from dengue type 2 virus; three additional distinct dengueserotypes have been recognized. Therefore, “80% E” also refers to thecorresponding peptide region of the envelope protein of these serotypes,and to any naturally occurring variants, as well as correspondingpeptide regions of the envelope (E) protein of other flaviviruses. Forexample, serotypes of dengue virus such as: DEN-1; DEN-2; DEN-3; andDEN-4, as well as serotypes of: Japanese encephalitis virus (JE),Tick-borne encephalitis virus (TBE), West Nile virus (WN), and thefamily prototype, Yellow fever virus (YF). The modifications made to the80% E products by addition of carboxy-terminal sequences encodingflexible linkers, leucine zipper domains, or four helix bundle domains,designed to enhance the dimerization of the 80% E molecules, aredescribed in detail below. All of these dimeric 80% E proteins areproduced from vectors containing the DNA encoding the flavivirus prM asa fusion with mature proteins resulting in secretion of the processed,mature dimeric 80% E proteins from which the prM protein has beenremoved.

[0052] Three basic approaches have been used to construct dimeric 80% Emolecules.

[0053] The first approach involves using tandem copies of 80% Ecovalently attached to each other by a flexible linker. As used herein,“Linked 80% E Dimer” refers in one instance to a polypeptide whichencodes DEN-2 80% E-GGGSGGGGSGGGTGGGSGGGSGG GG—DEN-2 80% E. The stretchof amino acids covalently linking the two copies of DEN2 80% E isdesigned to serve as a flexible tether allowing the two 80% E moleculesto associate in native head-to-tail dimeric orientation whilemaintaining their covalent attachment to each other. The sequencesdescribed in the present application represent the envelope protein fromdengue type 2 virus; three additional distinct dengue serotypes havebeen recognized. Therefore, “Linked 80% E Dimer” also refers to thecorresponding peptide region of the envelope protein of these serotypes,and to any naturally occurring variants, as well as correspondingpeptide regions of the envelope (E) protein of other flaviviruses. Forexample, serotypes of dengue virus such as: DEN-1; DEN-2; DEN-3; andDEN-4, as well as serotypes of: Japanese encephalitis virus (JE),Tick-borne encephalitis virus (TBE), West Nile virus (WN), and thefamily prototype, Yellow fever virus (YF).

[0054] It would be readily apparent to one of ordinary skill in the artto select other linker sequences as well. The present invention is notlimited to the specific disclosed linkers, but, to any amino acidsequence that would enable the two 80% E molecules to associate innative head to tail dimeric orientation while maintaining their covalentattachment to each other.

[0055] The second approach involves addition of a carboxy-terminalleucine zipper domain to monomeric 80% E to enhance dimerization betweentwo 80% E-leucine zipper molecules. Two versions of this approach havebeen adopted. One version includes a disulfide bond linking the leucinezipper domains resulting in a covalently linked dimer product, while theother is based on the non-covalent association of the leucine zipperdomains. As used herein “80% E ZipperI” refers in one instance to apolypeptide which encodes DEN-2 80%E-GGGSGGGGSGGGTGGGSGGGSPRMKQLEDKVEELLSKN YHLENEVARLKKLVGER. The first 22amino acids extending after the carboxy terminus of 80% E serve asflexible tether between 80% E and the adjacent leucine zipper domain.The leucine zipper domain is designed to dimerize with the identicalsequence from another 80% E Zipper molecule. The formation of anon-covalently linked leucine zipper will enhance the dimerization ofthe 80% E molecules, which may associate in native head to tailconformation by virtue of the flexible linker connecting the 80% Emolecules with the leucine zipper domain. The sequences described in thepresent application represent the envelope protein from dengue type 2virus; three additional distinct dengue serotypes have been recognized.Therefore, “80% E ZipperI” also refers to the corresponding peptideregion of the envelope protein of these serotypes, and to any naturallyoccurring variants, as well as corresponding peptide regions of theenvelope (E) protein of other flaviviruses. For example, serotypes ofdengue virus such as: DEN-1; DEN-2; DEN-3; and DEN-4, as well asserotypes of: Japanese encephalitis virus (JE), Tick-borne encephalitisvirus (TBE), West Nile virus (WN), and the family prototype, Yellowfever virus (YF).

[0056] It would be readily apparent to one of ordinary skill in the artto select other leucine zipper sequences as well. The present inventionis not limited to the specific disclosed leucine zipper sequences, butto any amino acid sequences that would enable the dimerization betweenidentical sequences from another 80% E Zipper molecule.

[0057] As used herein “80% E ZipperII” refers in one instance to apolypeptide which encodes DEN-2 80%E-GGGSGGGGSGGGTGGGSGGGSP-RMKQLEDKVEELLSKN YHLENEVARLKKLVGERGGCGG. Thefirst 22 amino acids extending after the carboxy terminus of 80% E serveas flexible tether between 80% E and the adjacent leucine zipper domain.The leucine zipper domain is designed to dimerize with the identicalsequence from another 80% E Zipper molecule. The leucine zipper domainof 80% E ZipperII ends in a GGCGG sequence which facilitates disulfidebond formation between the two leucine zipper helices. Thus, once theleucine zipper dimerizes, a disulfide bond forms between the two ends,resulting in a covalently linked dimer product. The formation of acovalently linked leucine zipper will enhance the dimerization of the80% E molecules, which may associate in native head to tail conformationby virtue of the flexible linker connecting the 80% E molecules with theleucine zipper domain. The sequences described in the presentapplication represent the envelope protein from dengue type 2 virus;three additional distinct dengue serotypes have been recognized.Therefore, “80% E ZipperII” also refers to the corresponding peptideregion of the envelope protein of these serotypes, and to any naturallyoccurring variants, as well as corresponding peptide regions of theenvelope (E) protein of other flaviviruses. For example, serotypes ofdengue virus such as: DEN-1; DEN-2; DEN-3; and DEN-4, as well asserotypes of: Japanese encephalitis virus (JE), Tick-borne encephalitisvirus (TBE), West Nile virus (WN), and the family prototype, Yellowfever virus (YF).

[0058] It would be readily apparent to one of ordinary skill in the artto select other leucine zipper sequences as well. The present inventionis not limited to the specific disclosed leucine sequences, but to anyamino acid sequences that would permit the dimerization with anidentical sequence from another 80% E Zipper molecule. Further, theordinary skilled artisan would readily be able to determine othersequences that would facilitate disulfide bond formation between the twoleucine zipper helices.

[0059] The final approach used to enhance dimerization of 80% E is theaddition of a helix-turn-helix domain to the carboxy terminal end of 80%E. The helix-turn-helix domain from one modified 80% E molecule willassociate with that of another to form a dimeric four-helix bundledomain. As used herein “80% E Bundle” refers in one instance to apolypeptide which encodes DEN-2 80%E-GGGSGGGGSGGGTGGGSGGGSP-GELEELLKHLKELLKG-PRK-GELEELLKHLKELLKGEF. Thefirst 22 amino acids extending after the carboxy terminus of 80% E serveas flexible tether between the 80% E domain and the helix-turn-helixdomain which follows. The formation of a non-covalently associatedfour-helix bundle domain will enhance the dimerization of the 80% Emolecules which may associate in the native head to tail conformation byvirtue of the flexible linkers connecting 80% E to the helix bundle. Thesequences described in the present application represent the envelopeprotein from dengue type 2 virus; three additional distinct dengueserotypes have been recognized. Therefore, “80% E Bundle” also refers tothe corresponding peptide region of the envelope protein of theseserotypes, and to any naturally occurring variants, as well ascorresponding peptide regions of the envelope (E) protein of otherflaviviruses. For example, serotypes of dengue virus such as: DEN-1;DEN-2; DEN-3; and DEN-4, as well as serotypes of: Japanese encephalitisvirus (JE), Tick-borne encephalitis virus (TBE), West Nile virus (WN),and the family prototype, Yellow fever virus (YF).

[0060] It would be readily apparent to one of ordinary skill of the artto select other amino acid sequences that would form the flexible tetherextending after the carboxy terminal of the 80% E and also comprising ahelix-turn-helix domain. The present invention is not limited to thespecific disclosed helix-turn-helix domains, but to any amino acidsequences that would enable the dimerization of one modified 80% Emolecule through a non-covalent association with a second modified 80% Emolecule. Further, the ordinary skilled artisan would readily be able todetermine other sequences that would facilitate such non-covalentassociation of helices.

[0061] Recombinant techniques provide the most practical approach forpractical large-scale production of these subunits for vaccine anddiagnostic purposes. However, to be efficacious these proteins mustundergo correct processing and assume a conformation similar to that ofnative flaviviral envelope protein. In order to achieve this, therecombinant production must be conducted in eucaryotic cells, preferablyDrosophila melanogaster cells. Other eucaryotic cells including yeast,mammalian cells such as Chinese hamster ovary cells, or additional typesof insect cells may also be used. However, to make a cost-effectivevaccine feasible, the dimeric 80% E products must be efficientlysecreted with correct processing and folding.

[0062] It has been found, as demonstrated herein below, thatparticularly efficient secretion of biologically active mature proteinis most easily achieved using the Drosophila melanogaster Schneider-2cell line. The expression of the dimeric products is driven by anefficient insect cell promoter (Drosophila metallothionein promoter) andsecretion is targeted using a eucaryotic secretion leader (human tissueplasminogen activator secretion leader) as well as the flaviviral prMprotein which contains the secretion signal for E. Other promoters andsecretion leaders can also be used. In general, the invention includesexpression systems that are operable in eucaryotic cells and whichresult in the secretion of dimeric truncated flaviviral envelopeproteins into the medium. Thus, useful in the invention are cells andcell cultures which contain expression systems resulting in theproduction and secretion of mature dimeric truncated flaviviral envelopeproteins.

[0063] The properly processed dimeric truncated E proteins are recoveredfrom the cell culture medium, purified, and formulated into vaccines.Purification and vaccine formulation employ standard techniques and arematters of routine optimization. Suitable formulations are found, forexample, in Remington's Pharmaceutical Sciences, latest edition, MackPublishing Company, Easton, Pa. In particular, formulations will includean adjuvant, such as alum or other effective adjuvant. Alternatively,the active ingredient and the adjuvant may be coadministered in separateformulations.

[0064] The active vaccines of the invention can be used alone or incombination with other active vaccines such as those containingattenuated or killed forms of the virus, or those containing otheractive subunits to the extent that they become available. The vaccinesmay contain only one subunit as an active ingredient, or additionalisolated active components may be added. Corresponding or differentsubunits from one or several serotypes may be included in a particularformulation.

[0065] To immunize subjects against flaviviral infection, the vaccinescontaining therapeutically effective amounts of the subunit areadministered to the subject in conventional immunization protocolsinvolving, usually, multiple administrations of the vaccine.Administration is typically by injection, typically intramuscular orsubcutaneous injection; however, other systemic modes of administrationmay also be employed. Less frequently used, transmucosal and transdermalformulations are included within the scope of the invention as areeffective means of oral administration. The efficacy of theseformulations is a function of the development of formulation technologyrather than the contribution of the present invention.

[0066] In addition to use in vaccines, the recombinant dimeric truncatedE proteins of the invention may be used as analytical reagents inassessing the presence or absence of anti-flaviviral antibodies insamples. Such uses include, but are not limited to, diagnosis ofinfection with any flavivirus, such as dengue, monitoring the responseto flaviviral infection, or use of immunoassays as part of standardlaboratory procedures in the study of the progress of antibody formationor in epitope mapping and the like. The antigens are employed instandard immunoassay formats with standard detection systems such asenzyme-based, fluorescence-based, or isotope-based detection systems.Preferably, the antigen is used coupled to solid support or in sandwichassays, but a multiplicity of protocols is possible and standard in theart.

[0067] Thus, the secreted dimeric proteins, linked 80% E dimer, 80% EZipperI, 80% E ZipperII, or 80% E Bundle, may be adsorbed onto solidsupport and the support then treated with a sample to be tested for thepresence of anti-flaviviral antibodies. Unbound sample is removed, andany bound antibodies are detected using standard detection systems, forexample, by treating the support with an anti-species antibody coupledto a detection reagent, for example horseradish peroxidase (HRP), withthe species specificity of the antibody determined by the sample beingtested. The presence of the HRP-conjugated antispecies antibody is thendetected by supplying a suitable chromogenic substrate. In addition, thedimeric proteins may be used to detect the presence or absence ofantibodies of various isotypes, including IgG and IgM isotypes by simplyaltering the specificity of the detecting antibodies. This may beparticularly significant as IgM antibodies to flavivirus are considereddiagnostic of a primary flaviviral infection. Alternatively, theanti-subunit or anti-flaviviral antibody may be adsorbed to the solidsupport and detected by treating the solid support with the recombinantdimeric proteins, either directly labeled, or labeled with an additionalantibody in a sandwich-type assay.

[0068] In another embodiment, this invention relates to diagnostic kitscomprising an antigen affixed to a solid support phase and animmunological detection system. The antigen of this invention is asecreted dimeric product used in conjunction with an immunologicaldetection system. The antigen includes the recombinant dimeric truncatedE protein in the form of a linked 80% E dimer or an 80% E ZipperI or an80% E ZipperII or an 80% E bundle. The solid support phase of thisinvention relates to any of those found in the art, including microtiterplates. The detection system of this invention relates to any of thosefound in the art including antihuman antibodies conjugated with adetectable enzyme label.

[0069] In the examples below, the expression, secretion, processing, andimmunogenicity of the secreted dimeric proteins, linked 80% E dimer, 80%E ZipperI, 80% E ZipperII, and 80% E Bundle are demonstrated. Theproducts are recombinantly produced as modified prM-80% E fusions whichare efficiently processed to remove the prM portion and secreted fromDrosophila cells. The secreted dimeric 80% E products are secreted athigh levels, up to 10 μg/ml in unselected cells, and they display acomplex pattern of glycosylation typical of mammalian and insect cellexpression systems. Furthermore, based upon reactivity withconformationally sensitive monoclonal antibodies, the secreted dimeric80% E products have native-like conformation and immunization of micewith dimeric 80% E, either crude or purified, induces a potentvirus-neutralizing immune response. The following examples are intendedto illustrate but not to limit the invention.

EXAMPLE 1 Construction of Expression Vector pMttD2prM2X80E for Secretionof Linked 80% E Dimer

[0070] DEN-2 strain PR159/S1 served as the source for all DEN-2 genesused in the invention. This strain has a small plaque,temperature-sensitive phenotype and differs from wild-type DEN-2 PR159strain at only one amino acid in the prM and E coding regions. A cDNAclone, pC8 (Hahn et al., 1988), derived from DEN-2 strain PR159/S1 wasused as starting material for generation of the subclones describedbelow. The sequence of the clone has been previously published (Hahn etal., 1988), however, complete sequencing of the pC8 clone, as well assubclones derived from pC8, in our laboratory has identified a number ofdiscrepancies with the published sequence. The complete nucleotidesequence and deduced amino acid sequence of the cDNA encoding the viralcapsid, prM, E, and NS 1 genes for PR159/S1 is included in FIG. 3. Shownin bold (and indicated with a *) at nucleotides 103, 1940, 1991, and2025 are corrections to the Hahn published sequence.

[0071] The pC8 cDNA clone was used to generate several subclonescritical for the construction of the dimeric 80% E clones included inthis invention. The first subclone encodes amino acids 1-395 of E (80%E). The primers D2E937p and D2E2121m, shown below, were used to amplifythe cDNA fragment extending from nucleotide 937 to 2121 andcorresponding to 80% E. These primers include convenient restrictionsites for cloning and the D2E2121m primer includes two stop codons afterthe 395th codon of E. The sequence of the primers is listed below withdengue sequence listed in uppercase letters and non-dengue sequenceslisted in lowercase letters.          Bgl II D2E937p 5′-cttctagatctcgagtacccgggacc ATG CGC TGC ATA GGA ATA TC -3′       XbaI   XhoI    SmaI     Met Arg Cys Ile Gly Ile Ser             Sal I D2E2121m 5′- gctctagagtcga cta tta TCC TTT CTT CPACCA G -3′        XbaI       END END Gly Lys Lys Phe Trp

[0072] The amplified 80% E cDNA fragment was digested with XbaI andcloned into the NheI site of pBR322 to obtain the plasmid p29D280E. Thecomplete nucleotide sequence of the clone was determined and a single,silent, PCR-induced mutation at nucleotide 2001 (AAC/Asn to AAT/Asn) wasidentified.

[0073] The portion of the genome that encodes prM and E was subclonedfrom pC8 using the Polymerase Chain Reaction (PCR). Oligonucleotideprimers were designed to amplify the region of the genome, nucleotides439 to 2421, corresponding to amino acids 1-166 of prM and 1-495 of Ewith convenient restriction sites engineered into the primers tofacilitate cloning. In addition the primer used to amplify the aminoterminus of the prM-E polyprotein includes a methionine codon (ATG)immediately preceding the first codon (phenylalanine) of the prM codingsequence. The sequence of the primers is listed below with denguesequence listed in uppercase letters and non-dengue sequences listed inlowercase letters.          Bgl II D2prM439p 5′-attctagatctcgagtacccgggacc atg TTT CAT CTG ACC ACA CGC -3′       XbaI   XhoI    SmaI     Met Phe His Leu Thr Thr Arg            Sal I  D2E2421m 5′- tctctagagtcga cta tta GGC CTG CAC CATAAC TCC -3′        XbaI       END END Ala Gln Val Met Val Gly

[0074] The PCR-generated prM100% E cDNA fragment was digested with therestriction endonuclease XbaI and ligated into the XbaI site ofpBluescript SK+(Stratagene, La Jolla, Calif.) to obtain the plasmidp29prME13. DNA sequence analysis of the PCR-generated cDNA cloneidentified two PCR-induced nucleotide differences between pC8 andp29prME13 in the prM-80% E coding region. The first mutation involves aT to C transition at nucleotide 1255 which is silent, and the secondchange involves an A to G transition at nucleotide 1117 which results inthe conservative amino acid substitution of a valine for an isoleucineat position 61 of E. This mutation was repaired by replacing an AflIIfragment containing the mutation with the corresponding AflII fragmentfrom pC8 encoding the correct sequence.

[0075] To generate a cDNA subclone representing prM80% E, a 794 bpBamHI-SalI fragment, representing the carboxy-terminal end of E, wasremoved from p29prME 13 and replaced with the 431 bp BamHI-SalI fragmentfrom p29D280E, encoding the carboxy-terminal end of 80% E. The BamHIsite is a naturally occurring site within the envelope cDNA, and theSalI site is an engineered site that immediately follows the stop codonsencoded by the PCR primers. The resulting truncated cDNA clone,pBsD2prM80E, was confirmed by restriction digestion and DNA sequenceanalysis to encode amino acids 1 through 166 of prM and 1 through 395 ofenvelope.

[0076] To engineer the Linked 80% E Dimer, cDNA encoding 80% E was PCRamplified in two “halves” from pC8 using primer/adapters that includethe flexible linker and a KpnI restriction endonuclease site tofacilitate ligation of the two halves. One half, designated PCR 1,encoded the carboxy terminus of the flexible linker and the aminoterminus of 80% E. The other half, designated PCR 2 encoded the carboxyterminus of 80% E and the amino terminus of the flexible linker. Thenucleotide sequences of the primers used to amplify the PCR 1 and PCR 2cDNAs are listed below. In each case, the cDNA fragments spanned anaturally occurring, unique BamHI site within the 80% E coding region.The strategy for generating and cloning the fragments is outlined inFIG. 4. The PCR products were digested with PstI and BamHI and clonedindividually into pUC plasmid vectors cut with the same two enzymes,resulting in plasmids pUC18PCR1 and pUC13PCR2 which were confirmed byDNA sequence analysis. The fragment encoding the amino terminus of 80% Ewas released from the pUC18PCR1 subclone by digestion with KpnI andcloned into pUC 13PCR2 linearized with KpnI to generate the clonepUC13PCR2+1 which encodes the carboxy terminus of 80% E—flexiblelinker—amino terminus of 80% E.

[0077] The primers used to generate cDNA fragment PCR1 were:       PstI  KpnI DI80E-2N 5′ AGT{overscore(CCTGCAGGTAC)}CGGTGGTGGTGGTTCTGGTGGTGGTTCTGGTGGTGGTATGCGTTGCATA a.a.sequence                T  G  G  G  G  S  G  G  G  S  G  G  G  M  R  C  IGGAATATCAAATAGG G  I  S  N  R D2E2007M 5′ CTATGATGATGTAGCTGTCTCC a.a.sequence      I  I  I  Y  S  D  G

[0078] The primers used to generate cDNA product PCR 2 were:          PstI  KpnI DI80E-1C 5′ GCTCAG{overscore(CTGCAGGTACC)}ACCACCAGAACCACCACCACCAGAACCACCACCACCTTTCTT a.a. sequence                  G  G  G  S  G  G  G  G  S  G  G  G  G  K  KGAACCAGTCCAGC  F  W  D  L D2E1642P 5′ GACACTGGTCACCTT a.a. sequence     T  L  V  T  F

[0079] To generate the sequence encoding prM plus the tandemly linkedcopies of 80% E, the cDNA fragment encoding carboxy terminus 80%E—flexible linker—amino terminus 80% E was released from the pUC13PCR2+1clone by digestion with BamHI. This BamHI fragment was then ligated intopBsD2prM80E digested with BamHI to yield pBsD2prM2X80E (FIG. 5).

[0080] To facilitate manipulations of the linked 80% E dimer expressionplasmid, we modified the Drosophila melanogaster expression vectorpMttbns (SmithKline Beecham). A XhoI site at nucleotide 885 was deletedby removing a 19 base pair BamHI fragment containing the XhoI site. Theresulting pMtt-Xho plasmid contained a unique XhoI site at nucleotide730 which precedes the SV40 polyadenylation signal and is useful forintroducing genes for expression studies. Plasmid pMtt-Xho was furthermodified to delete a KpnI site just upstream of the metallothioneinpromoter so that upon introduction of the linked 80% E dimer sequences,the KpnI site in the linker will be unique in the clone. To accomplishthis, the pMtt-Xho plasmid was digested with the restrictionendonuclease Acc65I. This enzyme has the same recognition sequence asKpnI but upon digestion results in a 5′ overhang which can be made flushupon incubation with Klenow fragment of DNA polymerase I anddeoxyribonucleotides. Thus digestion of pMtt-Xho with Acc65I followedwith Klenow treatment and ligation resulted in a plasmid, pMtt-HBG,which lacks the KpnI site (FIG. 6).

[0081] To introduce the linked 80% E dimer into the pMtt-HBG expressionplasmid, pBsD2prM2X80E was digested with BglII and SalI to release theprM-80% E—linker—80% E encoding fragment. This fragment was ligated intopMtt-HBG digested with BglII/SalI (FIG. 6). DNA sequence analysis of theresulting plasmid, pMttHBGD2prM2X80E, confirmed that the clone containedthe entire prM2X80E coding sequence but lacked the SV40 polyadenylationsignal. This clone is useful for introducing the oligonucleotidesencoding the leucine zipper and four-helix bundle domains (Examples 2,3, and 4) but is not useful for expression studies, as no poly A tail isassociated with low expression levels. To restore the poly adenylationsignal, the BglII/SalI fragment containing prM2X80E was removed from thepMttHBGD2prM2X80E clone and ligated into the pMtt-Xho plasmid digestedwith BglII and XhoI (FIG. 8). The resulting plasmid, pMttD2prM2X80E, wasused for transfection of Drosophila cells and expression studies.

EXAMPLE 2 Construction of Expression Vector pMttD2prM80EZipperI forSecretion of Non-Covalently Linked 80% E ZipperI

[0082] The plasmid pMttHBGD2prM2X80E was used as backbone for theintroduction of oligonucleotides encoding one half of the flexibleGly4Ser linker and the leucine zipper coiled coil helix. As illustratedin FIG. 7, this plasmid was digested with KpnI and SalI to remove afragment containing the carboxy-terminal half the flexible linker andthe second copy of 80% E. Four overlapping oligonucleotides, coding forthe carboxy-terminal half of the linker and leucine zipper helix wereannealed to each other, generating a KpnI site at the 5′ end and SalIsite at the 3′ end. The nucleotide and encoded amino acid sequence ofthe overlapping oligonucleotides are listed below. The annealed oligoswere ligated into the KpnI/SalI digested vector to generate theexpression plasmid, pMttHBGprM80EZipI. The identity of thepMttHBGprM80EZipI clone was confirmed by restriction digestion andlimited sequence analysis.

[0083] As described above however, the pMttHBGD2prM2X80E used asbackbone for this construct lacks the SV40 polyadenylation sequence.Therefore, the BglII/SalI fragment from pMttHBGprM80EZipI, encodingprM80% E ZipperI, was removed from the pMttHBGprM80EZipI plasmid andcloned into the BglII/XhoI digested pMtt-Xho vector to restore thedownstream polyadenylation signal (FIG. 8). The resulting plasmid,pMttD2prM80EZipI, was confirmed by restriction digestion and sequenceanalysis and used to transfect Drosophila cells for expression studies.Oligonucleotide Sequences:5′ GTACCGGCGGTGGCTCCGGCGGTGGCTCCCCCCGCATGAAGCAGCTGGAGGACAAGGTGGAGGAGCTGCT3′     GCCGCCACCGAGGCCGCCACCGAGGGGGGCGTACTTCGTCGACCTCCTGTTCCACCTCCTCGACGAa.a.   T  G  G  G  S  G  G  G  S  P  R  M  K  Q  L  E  D  K  V  E  E  L  LGT CCAAGAACTACCACCTGGAGAACGAGGTGGCCCGCCTGAAGAAGCTGGTGGGCGAGCGCTAATAGG 3′CAGGTTCTTCATGGTGGACCTCTTGCTCCACCGGGCGGACTTCTTCGACCACCCGCTCGCGATTATCCAGCT 5′  S  K  N  Y  H  L  E  N  E  V  A  R  L  K  K  L  V  G  E  R

EXAMPLE 3 Construction of Expression Vector pMttD2prM80EZipperII forSecretion of Covalently Linked 80% E ZipperII

[0084] The plasmid pMttHBGD2prM2X80E was used as backbone for theintroduction of oligonucleotides encoding one half of the flexibleGly4Ser linker and the leucine zipper coiled coil helix with a cysteineresidue close to the carboxy terminus. As illustrated in FIG. 7, thisplasmid was digested with KpnI and SalI to remove a fragment containingcarboxy-terminal half of the linker and the second copy of 80% E. Fouroverlapping oligonucleotides, coding for the carboxy-terminal half ofthe linker and cysteine-containing leucine zipper helix were annealed toeach other, generating a KpnII site at the 5′ end and SalI site at the3′ end. The nucleotide and encoded amino acid sequences of theoverlapping oligonucleotides are listed below. The annealed oligos wereligated into the KpnI/SalI digested vector to generate the expressionplasmid, pMttHBGprM80EZipII. The identity of the pMttHBGprM80EZipIIclone was confirmed by restriction digestion and limited sequenceanalysis.

[0085] As described above however, the pMttHBGD2prM2X80E used asbackbone for this construct lacks the SV40 polyadenylation sequence.Therefore, the BglII/SalI fragment from pMttHBGprM80EZipII, encodingprM80% E ZipperII, was removed from the pMttHBGprM80EZipII plasmid andcloned into the BglII/XhoI digested pMtt-Xho vector to restore thedownstream polyadenylation signal (FIG. 8). The resulting plasmid,pMttD2prM80EZipII, was confirmed by restriction digestion and sequenceanalysis and used to transfect Drosophila cells for expression studies.

[0086] Oligonucleotide Sequences: Oligonucleotide Sequences:5′ GTACCGGCGGTGGCTCCGGCGGTGGCTCCCCCCGCATGAAGCAGCTGGAGGACAAGGTGGAGGAGCTGCT3′     GCCGCCACCGAGGCCGCCACCGAGGGGGGCGTACTTCGTCGACCTCCTGTTCCACCTCCTCGACGAa.a.   T  G  G  G  S  G  G  G  S  P  R  M  K  Q  L  E  D  K  V  E  E  L  LGTCCAAGAACTACCACCTGGAGAACGAGGTGGCCCGCCTGAAGAAGCTGGTGGGCGAGCGCGGCGGTTGCGGCGGCAGGTTCTTCATGGTGGACCTCTTGCTCCACCGGGCGGACTTCTTCGACCACCCGCTCGCGCCGCCAACGCCGCC  S  K  N  Y  H  L  E  N  E  V  A  R  L  K  K  L  V  G  E  R  G  G  C  G  GTTAATAGG 3′ AATTATCCAGCT 5′

EXAMPLE 4 Construction of Expression Vector pMttD2prM80EBundle forSecretion of Non-Covalently Linked 80% E Bundle

[0087] The plasmid pMttHBGD2prM2X80E was used as backbone for theintroduction of oligonucleotides encoding one half of the flexibleGly4Ser linker and the helix-turn-helix domain. As illustrated in FIG.7, this plasmid was digested with KpnI and SalI to remove a fragmentcontaining the carboxy-terminal half of the linker and the second copyof 80% E. Four overlapping oligonucleotides, coding for thecarboxy-terminal half of the linker and helix-turn-helix domain wereannealed to each other, generating a KpnI site at the 5′ end and SalIsite at the 3′ end. The nucleotide and encoded amino acid sequences ofthe overlapping oligonucleotides are listed below. The annealed oligoswere ligated into the KpnI/SalI digested vector to generate theexpression plasmid, pMttHBGprM80EBundle. The identity of thepMttHBGprM80EBundle clone was confirmed by restriction digestion andlimited sequence analysis.

[0088] As described above however, the pMttHBGD2prM2X80E used asbackbone for this construct lacks the SV40 polyadenylation sequence.Therefore, the BglII/SalI fragment from pMttHBGprM80EBundle, encodingprM80% E Bundle, was removed from the pMttHBGprM80EBundle plasmid andcloned into the BglII/XhoI digested pMtt-Xho vector to restore thedownstream polyadenylation signal (FIG. 8). The resulting plasmid,pMttD2prM80EBundle, was confirmed by restriction digestion and sequenceanalysis and used to transfect Drosophila cells for expression studies.

[0089] Oligonucleotide Sequences: Oligonucleotide Sequences:5′ GTACCGGCGGTGGCTCCGGCGGTGGCTCCCCCGGCGAGCTGGAGGAGCTGCTGAAGCACCTGAAGGAG3′     GCCGCCACCGAGGCCGCCACCGAGGGGGCCGCTCGACCTCCTCGACGACTTCGTGGACTTCCTCa.a.    T  G  G  G  S  G  G  G  S  P  G  E  L  E  E  L  L  K  H  L  K  ECTGCTGAAGGGCCCCCGCAAGGGCGAGCTGGAGGAGCTGCTGAAGCACCTGAAGGAGCTGCTGAAGGGCGAGGACGACTTCCCGGGGGCGTTCCCGCTCGACCTCCTCGACGACTTCGTGGACTTCCTCGACGACTTCCCGCTCL  L  K  G  P  R  K  G  E  L  E  E  L  L  K  H  L  K  E  L  L  K  G  ETTCTAATAGG 3′ AAGATTATCCAGCT 5′  F

EXAMPLE 5 Expression and Secretion of Linked 80% E Dimer, 80% E ZipperI,80% E ZipperII, and 80% E Bundle from Drosophila melanogaster S2 Cells

[0090]Drosophila melanogaster Schneider-2 cells (S2; ATCC, Rockville,Md.) were cotransfected with each of the expression plasmids describedin detail above (pMttD2prM2X80Ef, pMttD2prM80EZipperI,pMttD2prM80EZipperII, or pMttD2prM80EBundle) and the selection plasmid,pCoHygro, at a weight ratio of 20:1 using the calcium phosphatecoprecipitation method (Wigler et al., 1979; Gibco BRL, Grand Island,N.Y.). The pCoHygro selection plasmid (van der Straten et al., 1989;SmithKline Beecham) encodes the E. coli hygromycin B phosphotransferasegene under the transcriptional control of the D. melanogaster copiatransposable element long terminal repeat and confers resistance tohygromycin B. Transfectants were selected for outgrowth in Schneider'smedium (Gibco BRL) supplemented with 10% fetal bovine serum (FBS;Hyclone) and 300 μg/ml hygromycin B (Boerhinger-Mannheim). Followingsignificant outgrowth, transfectants were plated at a cell density of2×10⁶ cell/m in serum-free IPL-41 medium supplemented with lipids,yeastolate, and Pluronic F68 (Gibco BRL) and induced with 200 μM CuSO₄.The media were harvested after 7 days of induction.

[0091] Proteins secreted into the culture medium were separated bySDS-PAGE, and analyzed by Coomassie blue staining and immunoprobing ofWestern blots with a polyclonal anti-DEN2 domain B (domain B correspondsto amino acids 296-395 of E). Under non-reducing conditions the expectedsizes for Linked 80% E Dimer, 80% E ZipperI, 80% E ZipperII, and 80% EBundle are 89.1 kD, 49.2 kD, 99.5 kD, and 49.5 kD respectively. Animmunoreactive band of appropriate molecular weight was detected inculture medium from all four constructs (FIG. 9A). This analysisconfirms that 80% E ZipperII, which was designed with cysteine residuesnear the carboxy terminal end of the leucine zipper alpha helices tofacilitate disulfide bond formation, is covalently dimerized by thedisulfide bond. This is in contrast to the non-covalently associated 80%E ZipperI and 80% E Bundle products which migrate as monomers underdenaturing but non-reducing conditions. Coomassie blue staining of thecrude media reveals a unique band which is plainly visible in the 80% EZipperI, 80% E ZipperII, and 80% E Bundle lanes (FIG. 9B). Comigratingbands of similar size make visualization of the Linked 80% E Dimer bandmore difficult. Based upon staining of protein standards we estimate theconcentrations of the dimeric proteins to be between 5 and 15 μg/mldepending on the construct and the growth conditions. Thus all fourdimeric 80% E proteins are expressed to high levels and efficientlysecreted from transfected Drosophila S2 cultures.

EXAMPLE 6 Secreted Dimeric 80% E Products are Glycosylated

[0092] Native dengue viral E is a glycoprotein displaying a complexpattern of glycosylation typical of mammalian- and insect cell-expressedproteins. Additional analyses of the secreted recombinant dimeric 80% Eproducts demonstrated that all four of the products are glycosylated.Crude media containing Linked 80% E Dimer, 80% E ZipperI, or 80% EBundle or purified 80% E ZipperII were denatured upon heat treatmentwith SDS and 2-mercaptoethanol prior to digestion with endoglycosidase H(EndoH) or peptide:N-glycosidase F (PNGase F). Digested and undigestedcontrol preparations were separated on SDS-PAGE gels and analyzed byCoomassie blue staining or Western blot analysis. Western blots probedwith polyclonal anti-DEN2 hyperimmune mouse ascites fluid (HMAF)demonstrate that all dimeric products are resistant to EndoH digestionbut sensitive to PNGase F digestion consistent with a complex pattern ofglycosylation (FIG. 10). Thus, the glycosylation pattern of all fourrecombinant dimeric 80% E products is similar to that of native dengueE. In addition, this blot demonstrates that under reducing conditions,80% E ZipperII runs as a monomer similar in size to 80% E ZipperI and80% E Bundle. This is again consistent with formation of a disulfidebond between the cysteines located near the carboxy-terminal end of theleucine zipper helices.

EXAMPLE 7 Recombinant Dimeric 80% E Products are Recognized byConformationally-Sensitive Monoclonal Antibodies

[0093] The reactivity of the recombinant dimeric 80% E products withconformationally-sensitive monoclonal antibodies (MAbs) was assessedusing indirect immunofluorescence assays (IFA). Transfected S2 cellswere plated onto slides and fixed with ice-cold acetone. The cells werethen treated with various polyclonal and monoclonal antibodies dilutedin PBS containing 20% FBS. After washing away unbound antibody, boundantibody was detected by reacting the cells with fluoresceinisothiocyanate-labeled goat anti-mouse immunoglobulin and observing on afluorescent microscope after excitation at 470 nm. Cells transfectedwith the Linked 80% E Dimer, 80% E Bundle, 80% E ZipperI, and 80% EZipperII were efficiently recognized by the conformationally sensitiveMAbs 9D12 and 4G2 (Henchal et al., 1992; Mason et al., 1989). Inaddition all transfectants were recognized by MAb 5A2 which binds to alinear epitope located in the domain B region of E (Megret et al.,1992). These data suggest that these recombinant, dimeric products areantigenically similar to native viral E and therefore may serve as auseful vaccine immunogen.

EXAMPLE 8 Induction of Dengue Virus Neutralizing Antibodies UponImmunization of Mice with Secreted Dimeric 80% E Produced by TransfectedS2 Cells

[0094] S2 cells expressing Linked 80% E Dimer, 80% E Bundle, 80% EZipperI, and 80% E ZipperII were cultured in serum-free medium(supplemented IPL-41; Gibco BRL) and induced by addition of CuSO₄ to afinal concentration of 0.2 mM in the culture medium (see example 5 formore detail on culture conditions). The cells were maintained ininducing medium for seven days prior to harvesting. The cells wereremoved by centrifugation at 1000×G in a Beckman TJ-6 refrigeratedcentrifuge and the media were filtered through a 0.2 μm celluloseacetate filter (Nalgene). The media containing the recombinant dimeric80% E products were concentrated approximately ten fold andbuffer-exchanged with PBS. The total protein concentration of the mediumwas determined using a dye binding assay (Biorad). Balb/c mice (JacksonLaboratories) were immunized intraperitoneally with 100 μg total proteinof each concentrated medium (of which only ˜5-10% was the dengueprotein) in Freund's complete adjuvant. The mice were boosted twice, attwo week intervals, with 50 μg of each medium in Freund's incompleteadjuvant. Ten days following the last boost the animals were sacrificedand their blood obtained for testing.

[0095] The sera from the immunized mice were tested for the presence ofantibodies which bind to recombinant DEN-2 80% E using an indirect ELISAassay. Briefly, plates were coated with purified, recombinant DEN-2 80%E, blocked with bovine serum albumin (BSA), and serial dilutions of themouse sera were then incubated with the coating antigen. Alkalinephosphatase-labeled goat anti-mouse IgG was used as the secondarydetecting antibody, and the color development upon addition of analkaline phosphatase chromogenic substrate was monitored. The ELISAtiter is the reciprocal of the highest dilution of serum which resultedin an optical density two-fold above background (reactivity of the serumagainst BSA only).

[0096] The sera were also tested for virus neutralizing antibodies usinga plaque reduction neutralization test (PRNT). In the PRNT assay, themouse sera were serially diluted in Eagles minimal essential medium(EMEM; BioWhittaker) supplemented with 10% FBS (Hyclone) and mixed with100 plaque forming units of Vero-adapted DEN-2 virus (from RobertPutnak, WRAIR). After allowing one hour for neutralization of the virus,the mixtures were plated onto susceptible monkey kidney monolayers (Verocells, from Robert Putnak, WRAIR) plated in EMEM containing 10% FBS in 6well tissue culture dishes (Costar). After allowing two hours for thevirus to bind, the cells were overlaid with 0.9% agarose (Fisher) inEMEM supplemented with 5% FBS. Viral cytopathic effect was allowed todevelop for 6-7 days and the viral plaques were stained with 0.012%neutral red (Sigma) in 1% agarose. The number of plaques in each clusterwere counted and compared to a no-serum viral control. The PRNT₈₀ titerwas the reciprocal of the highest dilution of serum which resulted in atleast 80% reduction in the number of plaques compared to the no-serumviral control. Results from the ELISA and PRNT assays are summarized inTable 1. All of the media induced a virus-binding and neutralizingresponse in the mice demonstrating that all of the dimeric 80% Eimmunogens are capable of functioning as efficient immunogens. TABLE 1Induction of Anti-DEN-2 Immune Response in Mice Immunized with CrudeMedia Containing Dimeric 80% E Products Mouse Number Immunogen ELISATiter PRNT₈₀ Titer 179-1 Linked 80% E Dimer 25,600 800 179-2 crudemedium 1600 10 179-3 100 μg 6400 1000 179-4 Freund's adjuvant 6400 400179-5 25,600 4000 180-1 80% E Bundle 1600 1000 180-2 crude medium 6400400 180-3 100 μg 6400 400 180-4 Freund's adjuvant 1600 200 180-5 64004000 181-1 80% E Zipperl 25,600 8000 181-2 crude medium 6400 200 181-3100 μg 6400 2000 181-4 Freund's adjuvant 6400 2000 181-5 1600 200 182-180% E Zipperll 25,600 800 182-2 crude medium 1600 100 182-3 100 μg 400100 182-4 Freund's adjuvant 1600 200 182-5 6400 1000 177-1 PBS <100 <10177-2 Iscomatrix <100 <10 177-3 Adjuvant <100 <10 177-4 <100 <10 177-5<100 <10

EXAMPLE 9 The Secreted, Recombinant Dimeric 80% E Products can beEfficiently Purified Using Immunoaffinity Chromatography

[0097] The conformationally sensitive MAb 9D12 has been previously usedin our laboratory to efficiently purify monomeric DEN-2 80% E. This MAbbinds to a conformational epitope in the domain B region (amino acids296-395) of DEN-2 E. MAb 9D 12 was covalently coupled to a HiTrap column(Pharmacia) and used to immunoaffinity-purify each of the recombinantdimeric 80% E molecules, Linked 80% E Dimer, 80% E ZipperI, 80% EZipperII, and 80% E Bundle. Crude media containing the products wasapplied to the column and unbound material removed by extensive washingwith phosphate-buffered saline (PBS). Bound material was eluted with 0.1M Glycine HCl pH 2.5 and immediately neutralized with 1.0 M Phosphate pH7.4. The products were concentrated and buffer exchanged into PBS priorto analysis on SDS-PAGE gels. Each of the products was efficientlypurified using this column (FIG. 11). In all cases the vast majority ofthe dimeric 80% E bound to the column and was efficiently eluted in arelatively small volume. Thus, this method offers an efficient means ofgenerating purified dimeric 80% E products for animal testing.

EXAMPLE 10 Induction of High Titer Dengue Virus-Neutralizing AntibodiesUpon Immunization of Mice with Purified, Secreted Dimeric 80% E

[0098] Culture media from S2 cells expressing Linked 80% E Dimer, 80% EBundle, 80% E ZipperI, and 80% E ZipperII, prepared as described inExample 8, were used as a source of antigen for additional mouseimmunization studies. Each of the products was purified usingimmunoaffinity chromatography (IAC) as described in Example 9.

[0099] Purified Linked 80% E Dimer, 80% E ZipperI, 80% E ZipperII, and80% E Bundle products were assayed using a quantitative Sandwich ELISAassay, SDS-PAGE analysis, and Western blotting. In the Sandwich ELISAassay MAb 9D12 was coated onto the plates, which were then blocked withBSA. Serial dilutions of a quantitated DEN-2 domain B standard or theproducts to be assayed were applied in triplicate to each well. Boundantigen was detected using a polyclonal rabbit anti-DEN-2 domain Bantibody and horseradish peroxidase-conjugated anti-rabbitimmunoglobulin. Chromogenic substrate for the horseradish peroxidase wasadded and the color development monitored. The absorbance generated bythe test antigen was compared to the standard curve and the amount ofantigen present in domain B equivalents is determined. To convert fromdomain B equivalents to dimeric 80% E, the weight ratio (˜4.5 for mostof the products), determined by comparing the relative molecular weightof the dimeric 80% E to domain B and dividing by the number of domain Bregions present in the dimeric 80% E product, was used. Each purifieddimeric product was quantitated using this assay for mouseimmunizations.

[0100] Balb/c mice (Jackson Laboratories) were immunized with 1 μg ofeach purified, secreted dimeric 80% E product. The immunizations weregiven subcutaneously using Iscomatrix (Iscotech) adjuvant. Twoimmunizations were given at 4 week intervals. Ten days following thefinal immunization the mice were sacrificed and their sera tested forvirus binding and neutralizing antibodies by ELISA and PRNT as describedin example 8. The results are summarized in Table 2. As is clearlyevident, all of the dimeric 80% E products induced a high-titer virusneutralizing response. These titers are higher than any titerspreviously reported in the literature and suggest that these dimeric 80%E products are exceptionally effective vaccine candidates. TABLE 2Induction of Anti-DEN-2 Immune Response in Mice Immunized with PurifiedRecombinant Dimeric 80% E Products Mouse Number Immunogen ELISA TiterPRNT₈₀ Titer 173-1 IAG-pure 102,400 4000 173-2 Linked 80% E Dimer102,400 8000 173-3 1 μg 102,400 8000 173-4 Iscomatrix 102,400 4000 173-5Adjuvant 102,400 4000 185-1 IAG-pure 102,400 32,000 185-2 80%E Bundle25,600 4000 185-3 1 μg 25,600 4000 185-4 Iscomatrix 25,600 16,000 185-5Adjuvant 102,400 2000 174-1 IAC-pure 6400 200 174-2 80% E Zipperl409,600 4000 174-3 1 μg 102,400 8000 174-4 Iscomatrix 102,400 16,000174-5 Adjuvant 102,400 8000 175-1 IAG-pure 102,400 8000 175-2 80% EZipperIl 25,600 2000 175-3 1 μg 102,400 16,000 175-4 Iscomatrix 102,4008000 175-5 Adjuvant 102,400 4000 176-1 IAG-pure 102,400 4000 176-2 80% E102,400 16,000 176-3 1 μg 25,600 8000 176-4 Iscomatrix 25,600 4000 176-5Adjuvant 102,400 4000 177-1 PBS <100 <10 177-2 Iscomatrix <100 <10 177-3Adjuvant <100 <10 177-4 <100 <10 177-5 <100 <10

EXAMPLE 11 Dose Response of Mice Immunized with Purified, SecretedRecombinant Dimeric Dengue 2 Virus Proteins

[0101] Culture media from S2 cells expressing dengue 2 virus (DEN-2) 80%E monomer, Linked 80% E Dimer, DEN-2 80% E Bundle, and DEN-2 80%ZipperII were used as source for the antigens. Each of the products waspurified using immunoaffinity chromatography as described in Example 9.The products were quantitated by ultraviolet spectroscopy. Balb/c micewere immunized by subcutaneous injection with 10, 1, or 0.2 μg of therespective recombinant products in 110 μg Iscomatrix adjuvant(Iscotech). Two immunizations were given at 4 week intervals. Ten daysfollowing the final immunization the mice were sacrificed and their seratested for virus neutralizing antibodies by PRNT test. The results aresummarized in Table 3. As is clearly evident, all of the recombinantproducts induced a high-titer virus neutralizing response even at verylow antigen doses. No statistically significant difference could bedetected between the groups. TABLE 3 Induction of Anti-DEN-2 ImmuneResponse in Mice Immunized with Purified Recombinant Dimeric orMonomeric 80% E Products Geometric Mean of PRNT₈₀ Titer Antigen 10 μgDose 1 μg Dose 0.2 μg Dose DEN-2 Linked 6355 2828 2766 80% E DimerDEN-280% E 6498 3732 1206 Zipperll DEN-2 80% E 9190 3482 777 BundleDEN-280% E 10,556 3031 1293 Monomer

EXAMPLE 12 Dimeric and Monomeric DEN-2 Recombinant 80% E Proteins Inducea Protective Response in Suckling Mice

[0102] Ten to 13 day old Balb/c mice were immunized by subcutaneousinjection with either 1 or 5 μg or immunoaffinity purified recombinantDEN-2 80% E monomer, Linked 80% E Dimer, 80% E ZipperII, or 80% E Bundlein 2 μg IscoMatrix. A second equivalent dose was administered two weekslater. One week following the final dose the mice were challenged byintracranial injection with 100 LD₅₀ of DEN-2 virus New Guinea C strainadapted for growth in mice. Morbidity and mortality was monitored for 17days post-challenge. The results are summarized in FIG. 12. Allimmunogens, at both 1 and 5 μg doses, resulted in complete protection ofthe suckling mice, demonstrating that the dimeric antigens induce potentprotective responses in mice.

EXAMPLE 13 Dimeric DEN-2 Antigens Induce Virus Neutralizing andProtective Responses in Primates

[0103] Groups of three rhesus monkeys were immunized with three doses of30 μg each of immunoaffinity purified DEN-2 80% E monomer, Linked 80% EDimer, 80% E ZipperII, or 80% E Bundle in 50 μg IscoMatrix adjuvant. Thedoses were administered subcutaneously on day 0, day 34, and day 120 ofthe study. Approximately one month following the final vaccination themonkeys were challenged by subcutaneous injection with 10⁴ pfu of liveDEN-2 virus (strain SI 6803). Control animals included animalsinoculated with live-attenuated DEN-2 Virus (PDK-50) or saline.Neutralizing antibody responses were monitored throughout the course ofthe experiment and are summarized in Table 4 below. In addition,protection from viral replication post-challenge was monitored bydetermining the level of virus in the blood for eleven dayspost-challenge. The results of the viremia assays are summarized inTable 5 below. In all cases a potent virus neutralizing response wasinduced by the vaccination schedule. In addition, significant protectionfrom viral challenge compared to monkeys immunized with saline wasobserved in all monkeys except one (FEV). TABLE 4 Virus neutralizingResponse in Monkeys Immunized with Recombinant DEN-2 80% E Dimers andMonomer Monkey Day 0 Day Day 34 Day Day Day 120 Day 153 ID ImmunogenVaccine 15 Vaccine 64 90 Vaccine Challenge Day 184 FEV 30 μg DEN-2 <1070 80 640 200 145 720 11,660 FKB Linked 80% E <10 60 40 1230 460 4156310 44,100 EKH Dimer <10 <10 55 1670 270 250 4060 14,310 Iscomatrix FJP30 μg DEN-2 <10 10 <10 950 260 120 3690 57,690 GPC 80% E Monomer <10 <10<10 630 205 130 3100 23,265 HTX Iscomatrix <10 <10 <10 540 160 150 16801290 HTB 30 μg DEN-2 <10 <10 30 950 150 130 3350 53,430 HTH 80% EZipperII <10 10 40 1180 250 180 3415 31,625 HPF Iscomatrix <10 115 20215 105 110 1525 11,810 HTF 30 μg DEN-2 <10 <10 15 70 80 90 2415 22,105GHF 80% E Bundle <10 10 95 1850 1110 665 10,595 18,835 GXD Iscomatrix<10 15 25 215 85 70 1060 16,260 GJK Saline <10 <10 <10 <10 <10 <10 <10380 HVA Iscomatrix <10 <10 <10 <10 <10 <10 <10 1080 FEB DEN-2 <10 <10<10 <10 <10 <10 <10 2510 GXJ PDK-50 <10 975 310 190 245 310 310 415 HOGVaccine <10 40 70 155 145 100 75 1180 GEG <10 65 5110 975 1450 1800 18251600

[0104] TABLE 5 Viremia in Vaccinated Monkeys Post-Challenge with LiveDEN-2 Virus Viremic Days Vaccine Animal 1 2 3 4 5 6 7 8 9 10 11 30 μgDEN-2 FEV 0 0 0 F F + + F 0 0 0 Linked 80% E FKB 0 0 0 0 0 0 +0 0 0 0Dimer EKH 0 0 0 0 0 0 0 0 0 0 0 Iscomatrix 30 μg DEN-2 FJP 0 0 0 0 + F +0 0 0 0 80% E GPO 0 0 0 0 F + 0 0 0 0 Monomer HTX 0 0 0 0 0 0 0 0 0 0 0Iscomatrix 30 μg DEN-2 HTB 0 0 0 0 0 0 + 0 0 0 0 80% E ZipperII HTH 0 00 0 0 0 0 0 0 0 0 Iscomatrix HPF 0 0 0 0 + F + 0 0 0 0 30 μg DEN-2 HTF 00 0 0 0 0 0 0 0 0 0 80% E Bundle GHF 0 0 0 0 0 + + 0 0 0 0 IscomatrixGXD 0 0 0 0 0 0 0 F + + 0 Saline GJK + + + + + + 0 0 0 0 0 IscomatrixHVA 0 0 + + + + + + 0 0 0 FEB 0 + + F + + F 0 0 0 0 DEN-2 GXJ 0 0 0 0 00 0 0 0 0 0 PDK-50 HOG 0 0 0 0 0 0 0 0 0 0 0 Vaccine GEG 0 0 0 0 0 0 0 00 0 0

EXAMPLE 14 Construction and Expression of Dimeric Form of Dengue 4 80% E

[0105] While the DEN-2 80% E monomer and dimer forms are both verypotent immunogens, the monomeric form of DEN-4 80% E is a much lesspotent immunogen. Therefore, a dimeric form (ZipperII form) of DEN-4 80%E was constructed to examine whether the dimeric form exhibits enhancedimmunogenicity. To construct the ZipperII form, the plasmidpMttD4prM80Ef.3+G.13, which encodes full-length prM and first 395 aminoacids of DEN-4 E, and pMttD2prM80E ZipperII which encodes full-lengthprM, the first 395 amino acids of DEN-2 E, the flexible linker andZipperII sequence described in detail in Example 3 were used astemplates. The 3′ end of DEN-4 80% E was PCR amplified from thepMttD4prM80Ef.3+G.13 template using an internal DEN-4 primer(P48D4E1435p; 5′-CCAGGTCACCATGGGTAG), corresponding to nucleotides1435-1452 of DEN-4, as positive strand primer and a negative strandprimer which corresponds to the last amino acids of DEN-4 80% E and thencontinues in frame to contain the 5′ end of the flexible linker up toand including the KpnI site (P64D4ZII-M;5′-ACCACCACCACCAGAACCACCACCCCCTTTCCTGAACCAATGGAGTG). The 3′ portion ofthe flexible linker (up to and including the KpnI site) and the ZipperIIsequence were PCR amplified from the pMttD2prM80E ZipperII templateusing the positive strand primer P64D4ZII-P (5′-TCAGGAAAGGGGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTACC) and the negative strand primer which bindswithin the pMttΔXho vector downstream of the SalI site (P64MTT1084-M;5′-ATACCGCAAGCGACAGGCCG). The resultant PCR product contains the secondhalf of the linker (including the KpnI site), the ZipperI sequence, thestop codons at the end of the ZipperII sequence, and the pMttΔXhosequence including the SV40 polyadenylation signal up to the SalI site.

[0106] The two PCR products contain an overlap which was utilized in anoverlap extension reaction to generate a single product of full-length.Briefly, the two PCR products were mixed together, heated and allowed toanneal to each other. Ten cycles of heating and slow annealing in thepresence of Taq DNA polymerase were conducted. Primers P48D4E1435p andP64MTT1084-M (positive strand primer from the DEN-4 80% E reaction withthe minus strand primer from the ZipperII reaction) were then added andstandard PCR amplification conducted. The full-length product wasdigested with SacI and SalI and ligated into pMttD4prM80Ef.3+G.13digested with SacI and SalI. Plasmid DNA from two independent bacterialtransformants, pMttD4prM80EZipII.1 and pMttD4prM80EZipII.2, wasconfirmed by restriction digestion and limited sequence analysis.

[0107] The expression plasmids were cotransfected into S2 cells usingthe calcium phosphate coprecipitation method (Wigler et al., 1979; GibcoBRL, Grand Island, N.Y.). The pCoHygro selection plasmid encodes the E.coli hygromycin B phosphotransferase gene under the transcriptionalcontrol of the copia transposable element long terminal repeat.Transfectants were selected for outgrowth in Schneider's medium (GibcoBRL) supplemented with 10% fetal bovine serum (Hyclone) and 300 μg/mlhygromycin B (Boerhinger Mannheim). Following significant outgrowth,transfectants were plated at a density of 2×10⁶ cells/ml in serum-freeIPL-41 medium supplemented with lipids, yeastolate, and Pluronice F68(Gibco BRL) and expression induced with 200 μM CuSO₄. The media wereharvested after 7 days of induction. Analysis of the culture media onSDS-PAGE gels revealed secretion levels ranging from 5-10 mg/L of DEN-480% E ZipperII. The recombinant DEN-4 80% E ZipperII product waspurified from the culture medium using immunoaffinity chromatography asdescribed in detail in Example 9 except that the conformationallysensitive monoclonal antibody 4G2 was used in place of 9D12.

EXAMPLE 15 DEN-4 80% E ZipperII Induces a Potent Virus NeutralizingResponse in Mice

[0108] Groups of 10 each adult Balb/c mice were immunized with variousdoses of immunoaffinity purified DEN-4 80% E monomer or dimeric DEN-480% E ZipperII. Doses of 30, 10, 3, 1, or 0.3 μg were administered bysubcutaneous injection with 10 μg IscoMatrix adjuvant. A secondequivalent dose was administered 4 weeks later. Ten days following thesecond dose the animals were sacrificed and the virus neutralizingantibody response assayed. The results are summarized in Table 6. Theimmunogenic superiority of the dimeric DEN-4 80% E ZipperII antigencompared to the DEN-4 80% E monomer is clearly evident from this study.TABLE 6 Virus Neutralizing Antibody Response Induced by Monomeric andDimeric DEN-4 80% E Antigens Dose Geometric Mean Geometric Mean ofAntigen PRNT₅₀ Titer PRNT₅₀ Titer (μg) DEN-4 80% E Monomer DEN-4 80% EZipperll 30 728 1400 10 526 1609 3 278 1613 1 144 1472 0.3 28 1251

1. A vaccine for the protection of a subject against infection by aFlavivirus, wherein said vaccine comprises a therapeutically effectiveamount of a dimeric 80% E, said dimeric 80% E having been secreted as arecombinantly produced protein from Drosophila Schneider cells, wherein80% E represents the N-terminal 80% portion of the protein from residue1 to residue
 395. 2. The vaccine of claim 1 wherein said dimeric 80% Eis selected from the group consisting of: linked 80% E dimer; 80% EZipperI; 80% E ZipperII; and 80% E Bundle.
 3. The vaccine of claim 2wherein the linked 80% E dimer is a truncated envelope protein ofserotype DEN-1.
 4. The vaccine of claim 2 wherein the linked 80% E dimeris a truncated envelope protein of serotype DEN-2.
 5. The vaccine ofclaim 1 wherein the linked 80% E dimer is a truncated envelope proteinof serotype DEN-3.
 6. The vaccine of claim 1 wherein the linked 80% Edimer is a truncated envelope protein of serotype DEN-4.
 7. Amultivalent vaccine for the protection of a subject against infection bya Flavivirus, wherein said vaccine comprises a therapeutically effectiveamount of a first dimeric 80% E product of one flaviviral serotype; asecond dimeric 80% E product of a second flaviviral serotype; a thirddimeric 80% E product of a third flaviviral serotype; and a fourthdimeric 80% E product of a fourth flaviviral serotype; wherein alldimeric 80% E products have been secreted as recombinantly producedprotein from a Drosophila Schneider cell, wherein 80% E is theN-terminal 80% of the protein from residue 1 to residue
 395. 8. Avaccine of claim 7 wherein said dimeric 80% E products are envelopeproteins of serotypes selected from the group consisting of: DEN-1;DEN-2; DEN-3; and DEN-4.
 9. The vaccine of claim 1 wherein saidFlavivirus is a dengue virus.
 10. The vaccine of claim 2 wherein saidFlavivirus is a dengue virus.
 11. The vaccine of claim 7 wherein saidFlavivirus is a dengue virus.
 12. A method to protect a subject againsta Flavivirus, which method comprises administering to a subject in needof such protection an effective amount of the vaccine of claim 1,wherein said 80% E is the N-terminal 80% of the protein from residue 1to residue
 395. 13. A method to protect a subject against a Flavivirus,which method comprises administering to a subject in need of suchprotection an effective amount of the vaccine of claim 1, wherein said80% E is the N-terminal 80% of the protein from residue 1 to residue395.
 14. An immunogenic polypeptide comprising a dimeric 80% E, saiddimeric 80% E having been secreted as a recombinantly produced proteinfrom Drosophila Schneider cells, wherein 80% E represents the N-terminal80% of the protein from residue 1 to residue
 395. 15. The immunogenicpolypeptide of claim 14 wherein said dimeric 80% E is selected from thegroup consisting of: linked 80% E dimer, 80% E ZipperI; 80% E ZipperII;and 80% E bundle.
 16. The immunogenic polypeptide of claim 15 whereinthe linked 80% E dimer is a truncated envelope protein which is at leastone member selected from the group consisting of serotype DEN-1,serotype DEN-2, serotype DEN-3, and serotype DEN-4.
 17. An immunogeniccomposition for the protection of a subject against infection byFlavivirus comprising the immunogenic polypeptide defined in claim 14and a physiologically acceptable carrier.
 18. The immunogeniccomposition defined in claim 17 further comprising an adjuvant.
 19. Theimmunogenic polypeptide defined in claim 17 wherein said adjuvant isIscomatrix.
 20. An immunodiagnostic for the detection of Flaviviruscomprising the immunogenic polypeptide defined in claim
 14. 21. Amultivalent immunodiagnostic for the detection of Flavivirus comprisingat least two of the immunogenic polypeptides defined in claim 14 of atleast two flaviviral serotypes.
 22. A vector host recombinant DNAexpression system, which comprises: a) a Drosophila host cell; b) asuitable recombinant DNA expression vector; c) DNA encoding dimeric 80%E, operably linked and under the control of a suitable promoter; and d)said DNA encoding dimeric 80% E operably linked to a secretory signalleader sequence, wherein 80% E represents the N-terminal 80% portion ofthe protein from residue 1 to residue
 395. 23. The vector hostrecombinant DNA system of claim 22, wherein said dimeric 80% E isselected from the group consisting of: linked 80% E dimer; 80% EZipperI; 80% E ZipperII; and 80% E Bundle.
 24. A DNA sequence encodingthe immunogenic polypeptide defined in claim
 14. 25. An immunodiagnostickit for the detection of Flavivirus in a test subject comprising a) theimmunogenic polypeptide defined in claim 14; b) a suitable support phasecoated with dimeric 80% E; and c) labeled antibodies immunoreactive toantibodies from said test subject.
 26. An immunodiagnostic kit for thedetection of Flavivirus in a test subject comprising a) the multivalentimmunodiagnostic polypeptide defined in claim 21; b) a suitable supportphase coated with dimeric 80% E; and c) labeled antibodiesimmunoreactive to antibodies from said test subject.